Center for Biofilm Engineering
Abstract:
"Characterization of Phenotypic Changes in Pseudomonas putida in Response to Surface-Associated
Growth"
01-033 The formation of complex bacterial communities known as
biofilms begins with the interaction of planktonic cells with a surface. A
switch between planktonic and sessile growth is believed to result in
a phenotypic change in bacteria. In this study, a global analysis of
physiological changes of the plant saprophyte Pseudomonas putida
following 6 h of attachment to a silicone surface was carried out by
analysis of protein profiles and by mRNA expression patterns. Two-dimensional
(2-D) gel electrophoresis revealed 15 proteins that were up-regulated
following bacterial adhesion and 30 proteins that were
down-regulated. N-terminal sequence analyses of 11 of the
down-regulated proteins identified a protein with homology to the ABC
transporter, PotF; an outer membrane lipoprotein, NlpD; and five
proteins that were homologous to proteins involved in amino acid
metabolism. cDNA subtractive hybridization revealed 40 genes that
were differentially expressed following initial attachment of P.
putida. Twenty-eight of these genes had known homologs. As
with the 2-D gel analysis, NlpD and genes involved in amino acid
metabolism were identified by subtractive hybridization and found to
be down-regulated following surface-associated growth. The gene for
PotB was up-regulated, suggesting differential expression of ABC
transporters following attachment to this surface. Other genes that
showed differential regulation were structural components of flagella
and type IV pili, as well as genes involved in polysaccharide biosynthesis.
Immunoblot analysis of PilA and FliC confirmed the presence of
flagella in planktonic cultures but not in 12- or 24-h biofilms. In
contrast, PilA was observed in 12-h biofilms but not in planktonic
culture. Recent evidence suggests that quorum sensing by bacterial
homoserine lactones (HSLs) may play a regulatory role in biofilm
development. To determine if similar protein profiles occurred during
quorum sensing and during early biofilm formation, HSLs extracted
from P. putida and pure C12-HSL were added to 6-h planktonic
cultures of P. putida, and cell extracts were analyzed by
2-D gel profiles. Differential expression of 16 proteins was observed
following addition of HSLs. One protein, PotF, was found to be
down-regulated by both surface-associated growth and by HSL addition.
The other 15 proteins did not correspond to proteins differentially
expressed by surface-associated growth. The results presented here
demonstrate that P. putida undergoes a global change in
gene expression following initial attachment to a surface. Quorum
sensing may play a role in the initial attachment process, but other
sensory processes must also be involved in these phenotypic changes.
Sauer, K and A.K. Camper, "Characterization of Phenotypic Changes in Pseudomonas putida in Response to Surface-Associated Growth," J. Bacteriology, 183(22):6579-6589 (2001).
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